Project Summary Membrane proteins play essential roles in many cellular processes. The overall goal of the proposed research is to understand the physical basis the protein folding process and the biophysical basis of membrane protein structures. At present, neither of these is well understood for membrane proteins. Thermodynamically, our work addresses the critical role that hydrophobicity plays in membrane protein folds. In the previous granting period, we developed a novel hydrophobicity scale that measures side-chain transfer free energies from water to the membrane center using a real bilayer and a real, folded membrane protein. Based on this achievement, we now propose to test the generality of this scale (1) By measuring side-chain transfer free energies using distinct membrane protein scaffolds; (2) By determining how extent-of-burial in the bilayer modulates water to bilayer transfer free energies; and (3) By engineering of our protein scaffold for measurements as a function of pH to address how the energetic consequences of ionizable group mutations vary with charge state. Kinetically, we discovered in the previous grant period that E. coli lipid head groups may act as energetic potentials that sort membrane proteins away from the wrong (inner) membranes and towards the correct (outer) membrane locations. We propose in a fourth aim to dissect the biophysical basis for this sorting by determining the kinetic lifetimes and conformations and activation energies to folding induced by E. coli-containing lipid head groups.